All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
The field of epigenetics has lagged behind genetics due to the lack of robust assays to measure DNA methylation. DNA methylation sensitive restriction enzymes were used in the first attempts to interrogate specific CpG sites for methylation. The use of methylation sensitive enzymes with Southern blotting or PCR (polymerase chain reaction) allowed the investigation of a limited number of individual CpG sites that were targets of restriction enzymes. The field of epigenetics was improved by the advent of DNA methylation using bisulfite treatment. This induces a primary sequence change in the DNA based on the DNA methylation status. Unmethylated C is converted to U and then to T by subsequent PCR. 5 mC remains unchanged and is read as C by subsequent PCR amplification.
This sequence can be assessed by a number of different methods: direct Sanger sequencing (bisulfite sequencing), restriction digests (COBRA), methylated-sequence specific PCR (MSP), sequence specific real time PCR (MethyLight/quantitative MSP), nucleotide extension assays (MS-SNuPE), and Pyrosequencing. However, these methods are labor intensive and do not lend themselves to high throughput assays. Currently, array based methods to measure DNA methylation of more than one gene do exist, but these depend upon multiplex bisulfite-PCR or restriction digestion with methylation sensitive restriction enzymes.
Thus, there is a need in the art for systems and methods to detect DNA methylation and mutations that do not require the use of PCR or other DNA amplification procedures.